Dorman:P1 phage (lysogenic)

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'''Day 2'''
'''Day 2'''
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-Subculture 200 μL of overnight culture into 10ml of L in a 250mL flask.
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-Subculture 200 μL of overnight culture into 15ml of L in a 250mL flask.
-Grow the bacteria at 32°C to an OD<sub>600</sub> of ~0.15.   
-Grow the bacteria at 32°C to an OD<sub>600</sub> of ~0.15.   
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[''Once moved to 37°C, the phage will become lytic and lyse the cells.'']
[''Once moved to 37°C, the phage will become lytic and lyse the cells.'']
-
-After a half-hour, monitor OD<sub>600</sub> values every 10 minutes.  When the value stops increasing (or drops), the cells have started to lyse.  Add several drops of chloroform, swirl, and leave them on the bench for 10 minutes.
+
-After a half-hour, monitor OD<sub>600</sub> values every 15 minutes.  When the value stops increasing (or drops), the cells have started to lyse.  Add several drops of chloroform, swirl, and leave them on the bench for 10 minutes.
''[It is important to stop the process with chloroform as soon as possible after lysis.  While subtle, there are visual changes to the culture after lysis.  It is still cloudy, but uniform- not the swirls typical of healthy growing cells.  In addition, the culture is more viscous, causing bubbles rise in the liquid  more slowly than before lysis.]''
''[It is important to stop the process with chloroform as soon as possible after lysis.  While subtle, there are visual changes to the culture after lysis.  It is still cloudy, but uniform- not the swirls typical of healthy growing cells.  In addition, the culture is more viscous, causing bubbles rise in the liquid  more slowly than before lysis.]''
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- Pour the supernatant into a fresh 15mL tube, add several drops of chloroform, and place in the fridge.
- Pour the supernatant into a fresh 15mL tube, add several drops of chloroform, and place in the fridge.
-
 
== Using the phage ==
== Using the phage ==

Revision as of 07:44, 24 March 2008

Contributed by Dan Stoebel

P1 cm is a temperature sensitive lysogenic generalized transducing phage of E. coli For a general introduction to the use of P1 for strain construction, see the page on P1vir. The critical thing to know about P1cm is that it forms lysogens at 32°C, but is lytic at 37°C. It also carries a chloramphenicol marker. This means that this phage cannot be used to transduce chloramphenicol markers in the E. coli chromosome (because there is no way to select for lysogens) or temperature sensitive mutants that require growth below 37°C. But aside from these cases, this technique gives me more reproducible results than using P1vir.

The following technique was taught to me by Dan Dykhuizen.


Making P1 lysogens

Day 1

Grow strains to be lysogenized overnight on L at 32°C

Day 2

-Add 100μL of bacteria and 10μL of 0.5M CaCl2 to a test tube.

-Add phage at a ratio of 1:1 (phage:bacteria) directly to the liquid.

-Incubate at room temperature for 30 min to allow the phage to absorb. It can go longer if neeeded.

- With a loop, streak onto LB/cam plates. If the P1 were at really low titers, also try plating 10<super>-2</super> dilutions.

-Grow overnight at 32°C (or over the weekend at room temperature.)

Making phage

Day 1

Grow lysogenic strains overnight in LB at 32°C

Day 2

-Subculture 200 μL of overnight culture into 15ml of L in a 250mL flask.

-Grow the bacteria at 32°C to an OD600 of ~0.15.

-Move the flasks to a 37°C shaking incubator. It is best to use a shaking water bath for this, as the rapidity of temperature change helps to induce lysis.

[Once moved to 37°C, the phage will become lytic and lyse the cells.]

-After a half-hour, monitor OD600 values every 15 minutes. When the value stops increasing (or drops), the cells have started to lyse. Add several drops of chloroform, swirl, and leave them on the bench for 10 minutes.

[It is important to stop the process with chloroform as soon as possible after lysis. While subtle, there are visual changes to the culture after lysis. It is still cloudy, but uniform- not the swirls typical of healthy growing cells. In addition, the culture is more viscous, causing bubbles rise in the liquid more slowly than before lysis.]

- Pour the liquid into a 15mL tube, balance all the tubes, and spin them in the centrifuge at 4000 rpms for 20 min.

- Pour the supernatant into a fresh 15mL tube, add several drops of chloroform, and place in the fridge.

Using the phage

For transductions, I use the same protocol as given on the P1vir page.

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